A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating

Abstract

Clathrin-coated pits assemble on a membrane and pinch off as coated vesicles. The released vesicles then rapidly lose their clathrin coats in a process mediated by the ATPase Hsc70, recruited by auxilin, a J-domain-containing cofactor. How is the uncoating process regulated? We find that during coat assembly small and variable amounts of auxilin are recruited transiently but that a much larger burst of association occurs after the peak of dynamin signal, during the transition between membrane constriction and vesicle budding. We show that the auxilin burst depends on domains of the protein likely to interact with lipid head groups. We conclude that the timing of auxilin recruitment determines the onset of uncoating. We propose that, when a diffusion barrier is established at the constricting neck of a fully formed coated pit and immediately after vesicle budding, accumulation of a specific lipid can recruit sufficient auxilin molecules to trigger uncoating.hsc70 ͉ endocytosis ͉ lipids

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

“…It was shown previously that the kinase GAK, which is necessary for CCV uncoating, was recruited shortly after the large GTPase dynamin to sites of CME [15,16]. Here, we found that GAK recruitment commenced at scission and peaked on average ,8 s thereafter, as predicted.…”

“…The kinase GAK is an established marker for CCV uncoating [15,16], and the GAK recruitment signature was the same for terminal and nonterminal scission events, for scission events at spot-like CCSs that formed de novo, and for scission events at different size and lifetime classes of CCSs.…”

“…Growing CCPs acquire cargo and invaginate via clathrin polymerization [4] and the coordinated action of curvature-inducing/sensing BAR [5] and F-BAR domain proteins [6,7], ENTH domain proteins [8], and possibly actin [9][10][11]. The neck of the deeply invaginated CCP is severed in a mechanism involving the large GTPase dynamin [12,13], and possibly a phosphoinositide (PI) phosphatase [14], to release a clathrin-coated vesicle (CCV), which uncoats through the action of GAK/auxilin [15,16].…”

Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages

“…Consequentially, this would guarantee selective retention of PI(4,5)P 2 at the plasma membrane and promote uncoating of vesicles immediately after fission (5). The SJ1-mediated change in phosphoinositide composition of the newly formed endocytic vesicle may favor uncoating by decreasing the affinity of clathrin adaptors with the membrane while simultaneously promoting the recruitment of auxilin, a cofactor for the ''uncoating ATPase'' HSC70 (39,40). The importance of SJ1 in clathrin uncoating is strongly supported by the accumulation of clathrin-coated vesicles in nerve terminals of SJ1 knockout mice (3,28).…”

Initiation de l’endocytose par vésicules de clathrine

“…Le détachement des vésicules de cla thrine de la membrane plasmique requiert l'action de la dynamine, une mécano-enzyme qui induit la fission de la base (« au cou ») de la vésicule [8]. Enfin, le désassemblage du manteau de clathrine de la vésicule nouvellement formée est assuré par l'ATPase Hsc70, recrutée par la protéine auxiline (Figure 1) [9][10][11]. La vésicule poursuit alors son chemin vers les endosomes précoces, et les composants de la machinerie du manteau de clathrine sont de nouveau disponibles pour un cycle.…”